Regioselective α-hydrolysis of amino acid diesters using pig liver esterase

ABSTRACT

The regioselective and chemoselective hydrolysis of an α-ester group of an amino acid diester using pig liver esterase enzyme (PLE) is disclosed. The amino acid diesters may be either N-protected or unprotected and the diester groups may be the same or different. In particular, the preparation of a number of γ-ester glutamates and β-ester aspartates are provided.

This application is a divisional of application Ser. No. 08/703,372filed Aug. 26, 1996 now U.S. Pat. No. 5,773,261.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to regioselective and chemoselectivehydrolysis of an α-ester group, of an amino acid diester using pig liveresterase enzyme (PLE). The amino acid diesters may be N-protected orunprotected and the ester groups may be the same or different.

2. Related Background Art

Throughout this application various publications are referenced byarabic numerals within parentheses (e.g., "Ref. 2"). Full citations forthese references may be found at the end of the specificationimmediately preceding the claims. Disclosures of these publications intheir entireties are hereby incorporated by reference into thisapplication to more fully describe the state of the art to which thisinvention pertains.

PLE has been used as a catalyst for the synthesis of chiral moleculesvia enantioselective ester hydrolysis (Ref. 1). PLE has also been usedto catalyze the hydrolysis of cyclic meso diesters to prepare chiralhalf-esters (Ref. 2). Other uses of PLE have included the hydrolysis ofester groups of labile molecules in the synthesis of achiral molecules(Ref. 3).

Generally, acid-esters or half-esters may be prepared by any of thefollowing methods: the esterification or transesterification of diacidswith an alcohol in the presence of an acid catalyst (Refs. 5, 8a and 8b); the hydrolysis of a cyclic anhydride with an alcohol (Refs. 6); orthe hydrolysis of a diester with barium hydroxide (Ref. 7 and 9). It isoften difficult to obtain a pure product (i.e. the monoester), usingthese methods due to the formation of a mixture of products formedduring inefficient isolation procedures. The mixture of products mayinclude the monoester, the diester, and the diacid. Additionally, thesemethods have the disadvantage of tending to give low yields of themonoester product.

The prior art reports several attempts to prepare amino acid monoestersby the selective hydrolysis of amino acid diesters Refs 4 and 11. Steinet al. demonstrated that the hydrolysis of aspartate dimethyl esterusing PLE resulted in the hydrolysis of both ester groups, i.e. theα-ester group and the β-ester group (Ref. 4). The selectivity of theα-ester hydrolysis to the β-ester hydrolysis was found to be 98:2 forthe formation of the corresponding aspartate monoesters In contrast,both the (R)-aspartate diethyl ester and (S)-aspartate diallyl ester areconverted to their respective β-monoesters with PLE with completeregioselectivity hydrolysis of the α-ester group. In addition, it wasfound that the preferential hydrolysis for the α-ester position is foundto be partially reversed when the aspartate is N-protected as itsformamide. The selectivity for the N-protected aspartate was found to be55:45 for the α-ester hydrolysis to the β-ester hydrolysis.

PLE has also been used by Adachi et al. in studying the regioselectivehydrolysis of optically active unsymmetric diesters (Ref. 2h). Inparticular, Adachi et al. disclosed that the hydrolysis of dimethylaspartate and dimethyl glutamate using crude PLE resulted in formationof a mixture of products due to hydrolysis of both ester groups. Thehydrolysis of N-protected aspartates and N-protected glutamates usingPLE also resulted in the formation of a mixture of products. Adachi etal. also reported that for the N-protected amino acid diestershydrolysis was slower at the α-ester group than the β- or γ-estergroups. The faster hydrolysis of the N-protected aspartates andglutamates at the β- and γ-ester groups is believed to be due to theless crowded methyl ester group at these positions being preferablyaccommodated at the active site.

In addition to PLE other enzymes such as porcine pancreatic lipase (PPL)has been used for the regioselective hydrolysis of dialkyl amino acidesters (Ref. 10). Hydrolysis of N-protected dialkyl aspartates using PPLresulted in the regioselective hydrolysis of the β-ester group andformation of the corresponding N-protected α-ester aspartate. However,in the case of unprotected dimethyl aspartate and glutamate esters,selective hydrolysis with PPL was not observed.

Accordingly, the development of an effective method for theregioselective hydrolysis of both u-protected and unprotected amino aciddiesters remains a challenging problem.

SUMMARY OF THE INVENTION

The present invention relates to the regioselective and chemoselectivehydrolysis of an α-ester group of an amino acid diester using pig liveresterase enzyme (PLE). The amino acid diesters may be N-protected orunprotected and the diester group may be the same or different. Inparticular, the invention relates to a method for the preparation ofγ-ester glutamate which comprises reacting a diester glutamatecomprising an α-ester group and a γ-ester group with an amount of a pigliver esterase enzyme effective to hydrolyze selectively the α-estergroup and form the γ-ester glutamate.

Furthermore, this invention relates to a method for the preparation ofan N-protected β-ester aspartate which comprises reacting an N-protecteddiester aspartate comprising an α-ester group and a β-ester group withan amount of a pig liver esterase enzyme effective to hydrolyzeselectively the α-ester group and form the N-protected β-esteraspartate.

Additionally, the invention provides for a method for the preparation ofa β-ester aspartate or a γ-ester glutamate which comprises reacting thecorresponding mixed diester aspartate or glutamate with PLE.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the regioselective and chemoselectivehydrolysis of an α-ester group of an amino acid diester using pig liveresterase enzyme (PLE). The amino acid diesters may be N-protected orunprotected and the diester group may be the same or different. Inparticular, a method for the preparation of a γ-ester glutamate whichcomprises reacting a diester glutamate comprising an α-ester group and aγ-ester group with an amount of a pig liver esterase enzyme effective tohydrolyze selectively the α-ester group and form the γ-ester glutamate.

The invention provides for the method where the α-ester group and theγ-ester group of the γ-ester glutamate may be the same or different.Each of the α-ester group and the γ-ester group is an alkyl ester. Inpreferred embodiments, the α-ester group and the γ-ester group areindependently selected from the group consisting of CH₃ OCO--, C₂ H₅OCO--, (CH₃)₃ COCO-- and C₆ H₅ CH₂ OCO--.

In one embodiment of the invention, the diester glutamate has anN-protecting group. The invention provides for the method where theα-ester group and the γ-ester group of the N-protected γ-ester glutamatemay be the same or different. Each of the α-ester group and the γ-estergroup is an alkyl ester. In preferred embodiments, the α-ester group andthe γ-ester group are independently selected from the group consistingof CH₃ OCO--, C₂ H₅ OCO--, (CH₃)₃ COCO-- and C₆ H₅ CH₂ OCO--.

The invention also relates to a method for the preparation of anN-protected β-ester aspartate which comprises reacting an N-protecteddiester aspartate comprising an α-ester group and a β-ester group withan amount of a pig liver esterase enzyme effective to hydrolyzeselectively the α-ester group and form the N-protected β-esteraspartate. The α-ester group and the β-ester group may be the same ordifferent. Each of the α-ester group and the β-ester group is an alkylester. In preferred embodiments of the method the α-ester group and theβ-ester group are independently selected from the group consisting ofCH₃ OCO--, C₂ H₅ OCO--, (CH₃)₃ COCO-- and C₆ H₅ CH₂ OCO--.

The N-protecting groups for the methods described herein are selectedfrom the group consisting of alkyl or alkyl carbonyl. Preferred examplesof N-protecting groups are selected from the group consisting of CH₃ CH₂--, CH₃ CO--, C₆ H₅ CH₂ CO--, C₆ H₅ CH₂ OCO--, CH₃ CH₂ CO-- and (CH₃)₃COCO--.

The invention further relates to a method for the preparation of aβ-ester aspartate which comprises reacting a diester aspartatecomprising an α-ester group and a β-ester group which are different withan amount of a pig liver esterase enzyme effective to hydrolyzeselectively the α-ester group and form the β-ester aspartate. Each ofthe α-ester group and the β-ester group is an alkyl ester. In preferredembodiments of the method the α-ester group and the β-ester group areindependently selected from the group consisting of CH₃ OCO--, C₂ H₅OCO--, (CH₃)₃ COCO-- and C₆ H₅ CH₂ OCO--.

In the methods of the present invention the amount of pig liver esteraseenzyme effective to hydrolyze selectively the α-ester group ispreferably about 150 units to about 575 units, more preferably about 300units to about 400 units, and most preferably about 300 units.

All of the methods described herein may be performed in the presence ofa suitable buffer. The buffer is selected from the group consisting ofphosphate, imidazole, piperazine-N,N'-bis(2-ethanesulfonic acid)(PIPES), N-2-acetamidoiminodiacetic acid (ACES) and3-(N-morpholino)propanesulfonic acid (MOPS). In one preferredembodiment, the phosphate buffer is potassium. The presence of a bufferensures that the pH of the reaction medium is constant:. The α-esterhydrolysis reaction is carried out preferably at a pH range of about 6to about 8, and more preferably at a pH range of about 6.5 to about 7.5.Organic solvents can be also be added to the buffer to aid in substratesolubility. Examples of suitable solvents include acetone, methanol,ethanol, N,N'-dimethylformamide (DMF), tetrahydrofuran (THF) and thelike.

The hydrolysis of N-protected dialkyl esters of aspartic acid using PLEresults in 100% regioselective hydrolysis of the α-ester group andformation of the β-ester product, as illustrated in the following Scheme1: ##STR1## wherein R and R' are defined in Table 1.

Scheme 1. Reaction of N-protected aspartic acid diesters (Compound 1)with PLE.

Similarly, hydrolysis of N-protected and unprotected dialkyl esters ofglutamic acid using PLE results in 100% regioselective hydrolysis of theα-ester group and formation of γ-alkyl esters, as illustrated in Scheme2 below: ##STR2## wherein R and R' are defined in Table 1.

Scheme 2. Reaction of N-protected and unprotected glutamic acid diesters(Compound 3) with PLE.

The rate of hydrolysis is faster when the amino acid diester substrateis soluble in the reaction medium and the enzyme is immobilized. Whenthe amino acid diester substrate is present as a suspension, PLE alsoregioselectively hydrolyses the α-ester group.

In the case of mixed diester amino acid substrates, PLE regioselectivelyhydrolyses the α-ester position. Hydrolysis of N-protected andunprotected α-methyl β-t-butyl aspartate (5) and α-methyl γ-t-butylester glutamate (7) gives the corresponding α-acids, as illustrated inScheme 3 below: ##STR3## wherein R and R' are defined in Table 1.

Scheme 3. PLE catalyzed hydrolysis of mixed diesters of aspartic acid(Compound 5) and glutamic acid (Compound 7).

A summary of the regioselective α-ester hydrolysis reactions of a numberof diester aspartates and diester glutamates with PLE is provided inTable 1. The following abbreviations are used in the columns of Table 1:Sub=substrate, Conc.=concentration of the substrate, % Yld.=percentageyield of product, and MP° C.=melting point in degrees centigrade,dec=decomposition, and lit.MP=literature melting point in degreescentigrade.

                                      TABLE 1    __________________________________________________________________________    Summary of the regioselective hydrolysis of diester aspartates and    diester    glutamates with PLE (Ref. 17).                   Conc.                       Enzyme  Time                                  MP ° C.    Sub       R    R'     (mmol)                       (Units)                           % Yld.                               hrs                                  (lit. MP) (reference)    __________________________________________________________________________    1  CH.sub.3            H      5.11                       300 38  18.1                                  193-195 dec (195-196) (Ref. 4)    1  CH.sub.3            C.sub.6 H.sub.5 CH.sub.2 OCO                   4.98                       300 34  28.4                                  96-97 (96-98) (Ref. 13)    1  CH.sub.3            C.sub.6 H.sub.3 CH.sub.2 OCO                   2.63                       300 75  30.4                                  96-98 (96-98) (Ref. 13)    1  CH.sub.3            CH.sub.3 CH.sub.2                   2.52                       300 57  43.1                                  203-205 (205-207) (Ref.16)    1  CH.sub.3            C.sub.6 H.sub.5 CH.sub.2 CO                   2.57                       300 62  46.8                                  77-78 (Ref. 17)    1  CH.sub.3            C.sub.6 H.sub.5 CH.sub.2 CO                   1.79                       150 48  14.8                                  77-78 (Ref. 17)    1  CH.sub.3            CH.sub.3 CO                   2.50                       300 25  24.9                                  143-145 (144-145) (Ref. 14)    1  CH.sub.3 CH.sub.2            H      4.99                       300 72  13.5                                  204-206 (205-206) (Ref. 15)    1  (CH.sub.3).sub.3 C            H      0.65                       150 84  26.5                                  194-195 dec (198-199) (Ref. 12)    1  C.sub.6 H.sub.5 CH.sub.2            H      2.53                       150 50  16.5                                  203-205 dec (205-207) (Ref. 4)    1  C.sub.6 H.sub.3 CH.sub.2            C.sub.6 H.sub.5 CH.sub.2 OCO                   0.66*                       450 34  48.7                                  84-86 (85-86) (Ref. 3b)    3  CH.sub.3            H      5.02                       150 60  1.5                                  179-181 (180-182) (Ref. 8a)    3  CH.sub.3            H      26.00                       575 81  4.5                                  179-181 (180-182) (Ref. 8a)    3  CH.sub.3 CH.sub.2            H      4.96                       300 76  17.7                                  195-197 (195-198) (Ref. 8a)    3  (CH.sub.3).sub.3 C            H      0.66                       150 50  38.1                                  189-190 dec (190-191) (Ref. 12)    3  CH.sub.3            CH.sub.3 CH.sub.2 CO                   1.76                       300 90  11.1                                  oil    5  --   H      4.97                       450 50  4.5                                  193-195 dec (198-199) (Ref. 12)    5  --   (CH.sub.3).sub.3 COCO                   4.97                       450 10  24.3                                  oil    7  CH.sub.3            H      4.99                       300 55  9.3                                  189-190 (190-191) (Ref.12)    __________________________________________________________________________     (*With 5% methanol as a cosolvent)

This invention will be better understood from the Examples which follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention and no limitation of the invention is implied.

The unprotected dialkyl amino acid esters are commercially availablefrom Sigma Chemical Company, St. Louis, Mo. The N-acyl and N-alkyldialkyl amino exters are prepared according to the literature procedures(Refs. 18, 19 and 20).

EXAMPLE 1 General Procedure for preparation of N-protected amino acidmonoesters.

In a typical experiment, PLE (150-450 units, suspension in ammoniumsulfate; SIGMA® Chemical Company, St. Louis, Mo.) was added to asuspension or solution of N-protected amino acid diester (0.66-13 mmol)in 15 mL of 0.01 M phosphate buffer (pH 7) at room temperature. The pHvalue was kept at about 7 by the addition of 1N sodium hydroxide. Afterconsumption of 1 mole equivalent of base, the pH was adjusted to 9, thenthe aqueous phase was washed with chloroform (2×10 mL). The aqueousphase was acidified to pH 2 with 1 N hydrochloric acid, and extractedwith chloroform (2×15 mL). The organic phase was washed with water,dried over anhydrous magnesium sulfate, and evaporated under reducedpressure to yield the product (Table 1).

EXAMPLE 2 General Procedure for preparation of unprotected amino acidmonoesters.

PLE (150-450 units, suspension in ammonium sulfate; SIGMA® ChemicalCompany, St. Louis, Mo.) was added to a solution of unprotected aminoacid diester (0.66-26 mmol) in 15 mL of 0.01 M phosphate buffer (pH 7)at room temperature. The pH value was kept at about 7 by the addition of1N sodium hydroxide. After consumption of 1 mole equivalent of base, thepH was adjusted to 3-5 by the addition of concentrated hydrochloricacid, the mixture was diluted with 500 mL of alcohol (methanol, ethanolor 2-propanol) and the precipitated solid was filtered. The crudecompound was crystallized from aqueous alcohols to give pure amino acidmonoester (Table 1).

References

1. Ohno, M. and Otsuka, M., Organic Reactions, 1989, Vol. 37; p.1.

2. (a) Hultin, P. G. et al., J. Org. Chem., 1991, 56, 5375; (b) Toone,E. J. and Jones, J. B., Tetrahedron: Asymmetry, 1991, 2, 207; (c)Kobayashi, S. et al., J. Org. Chem., 1990, 55, 1169; (d) Metz, P.,Tetrahedron, 1989, 45, 7311; (e) Van der Eycken, J., et al., J. Chem.Soc., Chem. Commun., 1989, 306; (f) Sabbioni, G. et al., J. Org. Chem.,1987, 52, 4565; (g) Sabbioni, G. et al., J. Chem. Soc., Chem. Commun.,1984, 236; (h) Adachi, K. et al., Chima, 1986, 40, 311.

3. (a) Ineyama, T. et al., The 54th Annual Meeting, Chemical Society ofJapan, 1987; (b) Jongejan, J. A. and Duine, J. A., Tetrahedron Lett.,1987, 28, 2767; (c) Hazato, A. et al., Nippon Kagaku Kaishi 1983, 9,1390; (d) Chem. Abstr., 1984, 100, 120720q.

4. Stein, K. A. and Toogood, P. L., J. Org. Chem., 1995, 60, 8110.

5. Swann, Jr., S. et al., Org. Syntheses Coll. Vol. II, 1943, 276 andreferences cited therein.

6. March, J., Advanced Organic Chemistry, Third Ed. 1985, 347.

7. Durham, L. J. et al., J. Org. Syntheses Coll. Vol. IV, 1963, 635.

8. (a) Albert, R. et al., Synthesis, 1987, 635; (b) Saito, Y. andNagoya, T., Jpn. Pat. 77,128,321 1977; (Chem. Abstr., 1977, 88,105783c).

9. Prestidge, R. L. et al., J. Org. Chem., 1975, 40, 3287.

10. Guibe-Jampel, E. et al., J. Chem. Soc., Chem. Commun., 1987, 1080.

11. (a) Miyazawa, T et al., J. Chem. Soc., Chem. Commun., 1988, 1214;(b) Cohen, S. G. et al., Biochemistry, 1963, 2, 820; (c) Cohen, S. G.and Crossley, J., J. Am. Chem. Soc. 1964, 86, 4999; (d) Chen, S. T. andWang, K.-T., Synthesis, 1987, 581; (e) Pugniere, M. et al., Tetrahedron:Asymmetry, 1992, 3, 1015.

12. Roeske, R., J. Org. Chem., 1963, 28, 1251.

13. Oki, K.; Suzuki, K.; Tuchida, S.; Saito, T.; and Kotake, H., Bull.Chem. Soc. Jpn., 1970, 43, 2554.

14. Marchatti, E.; Mattalia, G.; Curatolo, F.; and Bergesi, G., Ann.Chim., 1967, 57, 624.

15. Wiecozorek, W.; Bukowska-Strzyzewska, M.; Olma, A.; Kaminski, Z. J.;and Leplawy, M. T., J. Crystallogr. Spectrosc. Res., 1991, 21, 107.

16. Liu, K.-C. and Wang, D., Arch. Pharmaz., 1975, 308, 564.

17. Characterization of all of the compounds was confirmed by meltingpoint (mp), infra red spectroscopy (IR), and nuclear magnetic resonancespectroscopy (NMR).

18. Bodanszky, M., "Principals of Peptide Synthesis" (Springer-Verlag),1984, 85.

19. Bodanszky, M. and Bodanszky, A., "The Practice of Peptide Synthesis"(Springer-Verlag), 1984.

20. Ohfune, Y.; Kurokawa, N.; Higuchi, N.; Saito, M.; Hashimoto, M.; andTanaka, T., Chemistry Letters, 1984, 441.

What is claimed:
 1. A method for the preparation of an N-protectedβ-ester aspartate which comprises:reacting an N-protected diesteraspartate comprising an α-ester group and a β-ester group with an amountof a pig liver esterase enzyme effective to hydrolyze selectively theα-ester group and form the N-protected β-ester aspartate.
 2. The methodof claim 1, wherein the α-ester group and the β-ester group may be thesame or different.
 3. The method of claim 2, wherein each of the α-estergroup and the β-ester group is an alkyl ester.
 4. The method of claim 3,wherein the α-ester group and the β-ester group are independentlyselected from the group consisting of CH₃ OCO--, C₂ H₅ OCO--, (CH₃)₃COCO-- and C₆ H₅ CH₂ OCO--.
 5. The method of claim 1, wherein theN-protecting group is an alkyl or alkyl carbonyl.
 6. The method of claim5, wherein the N-protecting group is selected from the group consistingof CH₃ CH₂ --, CH₃ CO--, C₆ H₅ CH₂ CO--, C₆ H₅ CH₂ OCO--, CH₃ CH₂ CO--and (CH₃)₃ CCO--.
 7. The method of claim 1, further comprising thepresence of a suitable buffer.
 8. The method of claim 7, wherein thebuffer is selected from the group consisting of phosphate, imidazole,piperazine-N,N'-bis(2-ethanesulfonic acid), N-2-acetamidoiminodiaceticacid and 3-(N-morpholino)propanesulfonic acid.
 9. The method of claim 8,wherein the phosphate buffer is potassium.
 10. The method of claim 1,wherein the reaction is carried out at a pH range of about 6 to about 8.11. The method of claim 10, wherein the reaction is carried out at a pHrange of about 6.5 to about 7.5.
 12. The method of claim 1, wherein theamount of pig liver esterase enzyme is about 150 units to about 575units.
 13. The method of claim 7, further comprising the presence of asuitable organic solvent.
 14. The method of claim 13, wherein theorganic solvent is selected from the group consisting of acetone,methanol, ethanol, N,N'-dimethylformamide, and tetrahydrofuran.